Gamificación en Ambientes B-learning

and molecular-phylogenetic analyses

Jie Wei&Derek Peršoh&Reinhard Agerer

Received: 29 July 2009 / Revised: 21 October 2009 / Accepted: 5 November 2009

#German Mycological Society and Springer 2009

Abstract Morphological and anatomical characters of four ectomycorrhizae with affinities to the genera Humaria,

Geopora, andTrichophaea of Pyronemataceae (Pezizomy- cetes, Ascomycota) on Chinese Pine (Pinus tabulaeformis) are described. The ectomycorrhizae are yellowish brown to brown, and have pseudoparenchymatous outer mantle layers and partially warty emanating hyphae with thick walls and without clamps. Intrahyphal hyphae are present, and no rhizomorphs are formed. The four ectomycorrhizae are distinguishable by differences in cell shape of outer mantle layers and the presence of cystidia. Ectomycorrhizae of a possibleHumariaspecies (Pinirhiza humarioides) lack cystidia and have irregularly inflated cells on the outer mantle layer that are connected with thin septa. The two ectomycorrhizae showing probable affinities to Geopora

species (“P. daqingensis” and “P. geoporoides”) possess row-like arranged cells in the outer mantle layer and cell heaps, and differ by the presence or absence of cystidia as well as by the structure of the inner mantle layers. Ectomycorrhizae likely having been formed by a Tricho- phaeaspecies (“P. trichophaeoides”) have oval to polygo-

nal cells and no cystidia. The possible taxa affiliations were assessed by molecular-phylogenetic analyses of the internal transcribed spacer (ITS) and partial large subunit (LSU) nrDNA. Morphological and anatomical characters are discussed against the background of the LSU phylogeny.

Keywords Anatomy . Ectomycorrhiza . Morphology . Molecular-phylogenetic analyses . Pyronemataceae

Introduction

The majority of taxa in Pyronemataceae have traditionally been considered as being saprotrophic (Perry et al. 2007), but a few ectomycorrhizal fungi were also reported in this family: Genabea, Genea, Geopora, Humaria, Pulvinula,

Sphaerosporella, and Trichophaea (Amicucci et al. 2001; Danielson 1984; Erős-Honti et al. 2008; Fujimura et al.

2005; Jakucs et al.,1998; Smith et al.2006; Tedersoo et al.

2006). While Geopora and Wilcoxina form E-strain mycorrhizae (ectendomycorrhiza) (Fujimura et al. 2005; Yu et al. 2001), Tarzetta catinus (Holmsk.) Korf & J.K. Rogers andGeopyxis carbonaria(Alb. & Schw.: Fr.) Sacc. have been hypothesized as ectomycorrhizal associates of

Fagus sylvatica L. (Tedersoo et al. 2006) andPicea abies

L. (Vrǻlstad et al.1998).

Ectomycorrhizal anatomy is little studied in Pyronema- taceae, with detailed morpho-anatomical descriptions being only available for species of Genea, Humaria, and

Tricharina. While most of the species form pseudoparen- chymatous outer mantle layers, as in Genea, Humaria,

Trichophaea, and Geopora (Erős-Honti et al. 2008; Tedersoo et al.2006), ectomycorrhizae (ECM) ofPulvinula

and Tricharina gilva (Boud. ex Cooke) Eckblad (later identified as Wilcoxina mikolae Chin S. Yang & H.E. Wilcox by Egger 1996) form plectenchymatous outer mantle layers (Amicucci et al. 2001; Ingleby et al.1990). Adistinction by ECM features is difficult, especially between Genea and Humaria, because the ECM of both genera share common morpho-anatomical features, like angular cells in the outer mantle, and emanating hyphae

Electronic supplementary material The online version of this article (doi:10.1007/s11557-009-0637-x) contains supplementary material, which is available to authorized users.

J. Wei (*):D. Peršoh:R. Agerer

Department Biology I, Division of Organismic Biology: Mycology, University of Munich,

Menzinger Str. 67, 80638 Munich, Germany e-mail: [email protected] Mycol Progress

being colorless and smooth when young and yellowish brown and warty when old (Erős-Honti et al. 2008; Tedersoo et al.2006). In addition, identifications of ECM of Genea or Humaria, which were merely based on morpho-anatomical features, remain questionable, hence studies combining morphological and molecular approaches were demanded for a state-of-the-art identifica- tion of these ECM (Erős-Honti et al.2008).

In the course of an investigation of the ECM commu- nities on Chinese Pine (Pinus tabulaeformis Carr.), we found four anatomotypes, all of which have pseudoparen- chymatous outer mantle layers, thick-walled, yellowish to brownish, warty emanating hyphae without clamps, and lack rhizomorphs. They are similar to some ECM previ- ously reported in Pyronemataceae. In this study, their morpho-anatomical features are described in detail and molecular-phylogenetic analyses are applied to unravel their phylogenetic position. This is the first report of ECM in Pyronemataceae on Chinese Pine.

Materials and methods

Specimen sampling, ECM morphology and anatomy Soil samples were collected in pure Chinese Pine forests at Helan Mountain (Yinchuan City, Ningxia Hui Nationality Autonomous Region, China) and at Daqing Mountain (Huhhot City, Inner Mongolia Autonomous Region, China) throughout 2 years. ECM systems were assigned to anatomo- types and described according to Agerer (1987−2008,1991). Anatomical studies are based on at least 5 ECM for each anatomotype, and drawings were performed with the aid of a Normarski interference contrast microscope (Standard 14; Zeiss West Germany) connected with a drawing tube. All drawings were made at a magnification of ×1,000. Reference specimens of the mycorrhizae are deposited in M (see Holmgren et al.1990).

DNA sequencing

One unramified end, previously fixed in CTAB, from each of the four morphotypes was used for DNA extraction following careful microscopical examination to ensure that the isolated DNA originates from the respective anatomo- type. DNA of ECM was extracted using the DNAeasy Plant Mini Kit (Qiagen, Hilden, Germany) as recommended by the manufacturer. The nuclear rDNA (nrDNA) ITS and LSU regions were amplified using the PCR primers ITS1F (Gardes and Bruns1993) and ITS4 (White et al.1990) and LROR and LR5 (Moncalvo et al.2000), respectively. The obtained PCR product was purified using the QIAquick protocol (Qiagen), and fragments were sequenced applying

the same primers as for the PCR. Sequencing was performed by the sequencing service of the Department Biology I (Ludwig-Maximilians-Universität, München) using BigDye Terminator Ready Reaction Cycles Sequenc- ing Kit v3.1.

Sequence analyses

The most similar sequences were searched for in UNITE (Kõljalg et al. 2005, http://unite.ut.ee/) and GenBank (http://www.ncbi.nlm.nih.gov/) using megablast (Zhang et al.2000). The 100 sequences most similar to each obtained LSU sequence were downloaded from GenBank. Dupli- cates, i.e. identical sequences found as closest relatives of different query sequences, were omitted. Using the software BioEdit v7.0.5 (Hall 2005), the sequences were automati- cally aligned. The alignment was revised manually and columns not alignable with certainty were excluded from the following analyses. A total of 209 unique LSU sequences were retained for further molecular phylogenetic analyses. RAxML (Stamatakis 2006) was used for search- ing the most likely tree and for mapping of the bootstrap support values (500 replicates) upon this tree. The GTRCAT model of substitution was applied for both analyses having Maximum Likelihood as optimality crite- rion. The most parsimonious trees were searched for by executing batch files generated by PAUPRat (Sikes and Lewis 2001) in PAUP* v4.0 (Swofford 2003), with weighting mode set to multiplicative. Twenty replicates and 500 iterations were conducted. A consensus tree was calculated of all trees with equal (minimal) length and the posterior probabilities were noted for each branch. The following positions, according to DQ220352 (Humaria hemisphaerica (F.H. Wigg.) Fuckel), were alignable with certainty throughout all 209 taxa included in the LSU alignment: 21−61, 75−95, 112−170, 185−407, 411−533, and 544−579. The ITS nrDNA sequence from “P. humar- ioides” was additionally aligned with 62 of the best matching sequences found by“megablast”. The alignment subjected to the molecular-phylogenetic analysis included the reliably alignable positions: 110−144, 277−286, 290−300, 302−401, 403−480, 505−513, and 515−527 (according to EU819538,H. hemisphaerica).

Results

Morpho-anatomical descriptions

“Pinirhiza humarioides”

Morphological characters (Fig. 1a) Mycorrhizal systems

up to 5 mm long, main axes 0.4–0.5 mm diam.,

dichotomous, ramification orders 0–2 (3), hydrophilic, short distance exploration type. Unramified ends mostly straight, cylindric, sometimes slightly inflated at very tips, 0.4–1.3 mm long, 0.4 mm diam., brown when young, dark brown to black when older; surface of unramified ends loosely woolly, covered with few soil particles, mantle not transparent.Emanating hyphae infrequent, brownish under dissecting microscope. Cystidia lacking. Rhizomorphs

lacking.Sclerotiaabsent.

Anatomical characters of mantle in plan views (Figs. 2 and3) Mantle surface(Fig.2a, b) formed by a very thin, at places incomplete, pseudoparenchymatous layer composed of inflated, often irregularly shaped cells arranged in rows, 3–10 µm diam., with very thick cell walls, (1) 3–5 (11) µm wide, septa small and thin, surface with many soil particles.

Outer mantle layer(Fig. 3a) pseudoparenchymatous with angular cells (mantle type L/M, according to Agerer

1987−2008, 1991; Agerer and Rambold 2004–2009), neighboring cells sometimes connected by small and thin septa like in mantle surface, cells membranaceously yellowish to brownish, with infrequent solitary cells filled with granular contents, surface smooth, variable in dimension, 8–23 µm long, 5–8.5 (12) µm wide, cell walls 0.5–1.8 (2.5) µm.Inner mantle layer (Fig. 3b) plectenchymatous, hyphal cells 3.5–5 µm diam., membranaceously yellowish.Very tip(apex of the tip) organized like remaining parts.

Anatomical characters of emanating elements (Figs. 1b and 4) Rhizomorphslacking. Emanating hyphae (Figs.1b

and 4) infrequent, originating from a hyphal cell of outer mantle, distal of ramification point always sinuous; septa simple and frequent, thinner than walls; walls thick, uneven; ramifications frequent, angle of ramification acute to rectangular, side-branches thinner in diameter than the main hypha; main hyphae 7–11 µm diam., up to 14 µm at the base, cell walls 2–2.5 µm; side-branches 3–5.5 µm diam., cell walls 0.5–1.5 µm; cells (15) 23–35 (45) µm long; surface of hyphae with alternating smooth and warty

Fig. 1 “Pinirhiza humarioides”:ahabit of ectomycorrhiza, with few emanating hyphae and soil particles, b emanating hyphae with partially distinctly warty and partially smooth surface, intrahyphal hyphae present; note the cylindric, sometimes capitate warts

Fig. 2 Surface layer of outer mantle of “Pinirhiza humarioides”:

a hyphal cells variable in shape, cells connected by thin septa and arranged in irregular rows,bwith more regularly-shaped hyphal cells like in outer mantle layer

portions, rough areas with cylindrical or capitate warts up to 1 µm long and up to 1.1 µm diam. at apex; tip of hyphae simple or ramified, often with some adhering soil particles; hyphae brownish when old, yellowish to colourless when young.Cystidialacking.Clamydosporeslacking.

Colour reactions with different reagents Preparations of mantle: Melzer’s reagent: n.r. (= no reaction); lactic acid:

n.r.; KOH: n.r.; FeSO4: n.r.

Reference specimen The mycorrhiza was collected in a pure Pinus tabulaeformis stand at Helan Mountain, Suyu- kou National Reserve located in Yinchuan City, Ningxia Hui Nationality Autonomous Region, China, myc. exc. and isol. by Jie Wei, 09.09.2008, JW 189d (in M). Sequences obtained: ITS (GQ281479) and LSU (GQ281475).

“Pinirhiza daqingensis”

Morphological characters (Fig. 5a) Mycorrhizal systems

3–5 (8) mm long, main axes 0.4–0.5 mm diam., dichoto- mous or irregularly ramified, ramification order 0–6, contact to short distance exploration type, hydrophilic.

Unramified ends straight, irregularly inflated, constricted between old and young parts, 1.0–3.5 mm long, 0.3–

Fig. 4 Emanating hyphae of “Pinirhiza humarioides”: angle of ramification ca.90°, one hyphal branch is thinner than the main hypha (arrow)

Fig. 3 Mantle layers of“Pinirhiza humarioides”:aouter mantle layer with angular cells, some of them connected by thin septa, cells arranged in rows,bplectenchymatous inner mantle layer

Fig. 5 “Pinirhiza daqingensis”:ahabit of ectomycorrhiza, surface of mantle partially densely short spiny,bdifferent developmental stages from roundish cells to cystidia,cawl-shaped cystidia on outer mantle layer together with some roundish cells arranged in patches

0.5 mm diam., younger parts yellowish brown, older parts reddish brownish, mantle not transparent, cortical cells not visible. Surface of unramified ends loosely short-spiny, cystidia not specifically distributed, concolorous to mantle.

Emanating hyphaeinfrequent. Rhizomorphslacking. Scle- rotianot observed.

Anatomical characters of mantle in plan views (Figs. 6 and 7) Outer mantle layers (Fig. 6a, b) pseudoparenchy- matous with angular cells and many roundish cells on the mantle surface, solitary or arranged in groups that can bear prominent cystidia (mantle type K, according to Agerer

1987−2008,1991; Agerer and Rambold2004–2009), some small areas of mantle slightly depressed; hyphal cells partially arranged in rows, sometimes also star-like struc- tures present; roundish cells 4–11 µm in diam., cell walls 0.5 µm thick, cells of the outer mantle layer 6–15 µm wide and 13–24 µm long; cells membranaceously yellowish to brownish, surface smooth. Inner mantle layers (Fig. 7) plectenchymatous with few angular cells, cylindric hyphae 6–8 µm diam., angular cells 12.5–25 µm long, 5.5–15 µm

wide, colourless and smooth.Very tip similar to remaining parts of the mantle.

Anatomical characters of emanating elements (Figs. 5b, c and 8) Rhizomorphs lacking. Emanating hyphae (Fig. 8)

Fig. 7 “Pinirhiza daqingensis”: plan view of inner plectenchymatous mantle layer

Fig. 6 Outer mantle layer of“Pinirhiza daqingensis”: asome cells forming heaps and cystidia,bplan view of outer mantle layer, hyphal cells arranged in rows (thickness of cell walls not shown)

Fig. 8 Emanating hyphae of “Pinirhiza daqingensis”with frequent simple septa and some adhering soil particles, warts and intrahyphal hyphae

infrequent, (3) 6.5–7.5 µm diam., cell walls 0.5 µm, hyphal cells short, distance of septa (9) 13–17 (20) µm, septa simple; hyphae frequently ramified, angle of ramification acute or sometimes ca. 90°; surface mucilaginous with many soil particles and warty, warts cylindrical, up to 1.5μm long and 0.5–1 µm wide; hyphae membranaceously yellowish; intrahyphal hyphae present.Cystidia(Fig.5b, c) very frequent (on very tip infrequent), bottle- to awl- shaped, with a strongly inflated base and a long torn-out neck, neck separated from inflated body by a septum, inflated body 10–11.5 µm diam. and with 0.5–1 µm thick walls, necks 2.5–3.5 µm wide and with 0.5 µm thick walls, cell wall at very tip thinner than at remaining parts; cystidia 21–54 (90) µm long, neck with 1–3 septa, membrana- ceously yellowish to brownish, some crystals present on surface.Chlamydosporeslacking.

Colour reactions with different reagents Preparations of mantle: Melzer’s reagent: n.r.; lactic acid: n.r.; KOH: n.r.; FeSO4: n.r.

Reference specimenThe mycorrhiza was collected in a pure Pinus tabulaeformis stand at Daqing Mountain, Guluban located in Huhhot city, Inner Mongolia Autono- mous Region, China, myc. exc. and isol. by Jie Wei, 07.26. 2007, JW 76a (in M). Sequences obtained: ITS (GQ281480) and LSU (GQ281476).

“Pinirhiza geoporoides”

Morphological characters (Fig. 9a) Mycorrhizal systems

dichotomous, with 0–3 orders of ramification, solitary or in few numbers, main axis 0.4–0.5 mm diam., contact to short distance exploration type, hydrophilic. Unramified ends

straight or bent, cylindric, not inflated, 0.5–1.8 mm long, 0.35 mm diam., yellowish brown, older parts dark brown; mantle not transparent, loosely woolly.Emanating hyphae

infrequent.Rhizomorphsabsent.Cystidialacking.Sclerotia

lacking.

Anatomical characters of mantle in plan views (Figs. 10 and11) Outer mantle layers(Fig.10a, b) pseudoparenchy- matous with angular cells, in some parts cells arranged in rows and in some parts forming heaps, and with few solitary, roundish, thick-walled (0.5 µm) cells (mantle type K, according to Agerer 1987−2008, 1991; Agerer and Rambold2004–2009), surface of mantle with a gelatinous matrix gluing many soil particles, roundish cells 5–7 µm in diam., other cells 12–23.5 µm long, and 6.5–13 µm wide, walls 0.3–0.5 µm thick, surface smooth, membranaceously brownish, plasmatically brownish when old, but colorless when young. Middle mantle layer (Fig. 11a) transitional

between pseudoparenchymatous with mostly angular cells and few irregularly shaped cells and short hyphal cells intermixed, some parts forming ring-like structures, cells membranaceously yellowish to brownish, 7–15 µm long and 6.5–11 µm wide.Inner mantle layers (Fig. 11b) plectenchymatous, hyphae ring-like arranged, cells 3– 3.5 µm wide, with some thicker hyphae up to 5.5 µm diam., cell walls thin, hyphae connected by anastomoses, membranaceously yellowish. Very tip like remaining parts of mantle.

Anatomical characters of emanating elements (Fig. 9b) Rhizomorphs lacking. Emanating hyphae (Fig. 9b) very infrequent, originating directly from an outer mantle layer cell, at the base not thicker than at other parts, 5–5.5 µm diam., cells (23) 27–45 (60) µm long, thick-walled, walls 1–1.5 µm, brownish, finely warty, simple septa, few soil particles adhering, intrahyphal hyphae present. Cystidia

lacking. Chlamydosporeslacking.

Fig. 9 “Pinirhiza geoporoides”: a habit of ectomycorrhiza, b

emanating hyphae originating from cells of outer mantle, surface partially warty, intrahyphal hyphae present

Colour reactions with different reagents Preparations of mantle: Melzer’s reagent: n.r.; lactic acid: n.r.; KOH: n.r.; FeSO4: n.r.

Reference specimenThe mycorrhiza was collected in a pure Pinus tabulaeformis stand at Daqing Mountain, Guluban located in Huhhot city, Inner Mongolia Autono- mous Region, China, myc. exc. and isol. by Jie Wei, 06.08.2008, JW 96a (in M). Sequences obtained: ITS (GQ281481) and LSU (GQ281477).

“Pinirhiza trichophaeoides”

Morphological characters (Fig.12a) Mycorrhizal systems

1.2–2.5 mm long, dichotomous, with 0–3 orders of ramification, solitary or in small numbers, main axes 0.3– 0.45 mm diam., short distance exploration type, hydrophil- ic. Unramified ends straight, cylindric, not inflated, 0.2–

1.8 mm long, 0.3–0.4 mm diam., reddish brown, very tips lighter, greyish, older parts dark brown or black; mantle not transparent, loosely to densely long-woolly. Emanating

hyphae frequent. Rhizomorphs absent. Cystidia lacking.

Sclerotialacking.

Anatomical characters of mantle in plan views (Figs. 13, 14) Outer mantle layers (Fig.13a) pseudoparenchymatous with angular cells and with oval to polygonal cells forming heaps, and also with solitary, small, roundish cells, 6–8 µm in diam., with 0.5–1 µm thick walls (mantle type K, according to Agerer1987−2008,1991; Agerer and Rambold

2004–2009), cells of outer mantle layer 9–23 µm long

and 4–15 µm wide, walls 0.5–1 µm; surface smooth, membranaceously brownish, plasmatically brownish when old, but colourless when young, mantle surface with many soil particles.Middle mantle layers(Fig.13b) pseudoparen- chymatous with angular cells, without special arrangement, cells membranaceously yellowish to brownish, 10–22 µm long and 5–12 µm wide. Inner mantle layers (Fig. 14) transitional between pseudoparenchymatous with epider- moid cells and plectenchymatous, without pattern, cells membranaceously yellowish to brownish, plasmatically brownish, cells 3.5–7 µm diam. Very tip like remaining parts of mantle.

Fig. 11 “Pinirhiza geoporoides”,:aplan view of middle mantle layer, cells of some parts arranged in rows,b plan view of inner mantle layer, plectenchymatous with ring-like structures

Fig. 10 Outer mantle layer of “Pinirhiza geoporoides”,: a some hyphal cells arranged in rows, surface of outer mantle with matrix and few adhering particles (shown only at the margins of the mantle piece),bplan view of outer mantle layer

Anatomical characters of emanating elements (Fig.12b–d) Rhizomorphs lacking. Emanating hyphae (Fig. 12b–d) frequent, originating directly from an outer mantle layer cell, 3.5–5 (8) µm diam., cells (18) 30–55 (65) µm long, cell walls 0.5–1 (1.5) µm; frequently ramified, angle of ramification ca. 90°; anastomoses open with a short or long bridge, anastomoses smooth; septa simple, thinner than walls; hyphae partially smooth and partially warty, often smooth near the origin; hyphae plasmatically brownish when old, membranaceously yellowish to colorless when young; very tip of hyphae simple, sometimes slightly swollen, often with adhering soil particles; intrahyphal hyphae present.Cystidialacking.Clamydospores lacking.

Colour reactions with different reagents Preparations of mantle: Melzer’s reagent: n.r.; lactic acid: n.r.; KOH: n.r.;

FeSO4: n.r.

Fig. 14 Plan view of inner mantle layer of “Pinirhiza tricho- phaeoides”

Fig. 13 “Pinirhiza trichophaeoides”,: a plan view of outer mantle layer, roundish and polygonal cells forming heaps, bplan view of middle mantle layer

Fig. 12 “Pinirhiza trichophaeoides”,: a habit of ectomycorrhiza, surface of mantle partially densely woolly,bemanating hyphae with partially warty and partially smooth surface and with some adhering soil particles, anastomoses open and with a long bridge,cemanating hyphae, anastomosis open and with a short bridge, d intrahyphal hyphae

Reference specimenThe mycorrhiza was collected in a pure Pinus tabulaeformisstand at Helan Mountain, Suyu- kou National Reserve located in Yinchuan City, Ningxia Hui Nationality Autonomous Region, China, myc. exc. and isol. by Jie Wei, 16.08.2007, JW 44a (in M). Sequences obtained: ITS (GQ281482) and LSU (GQ281478).

Sequence analyses

The results of the Blast searches among the sequences deposited in GenBank and in UNITE were ambiguous for both loci, i.e. a reliable assignment of any sequence to a certain taxon was not possible. Therefore, these results are not discussed in detail here, but the results of the molecular- phylogenetic analyses of all best matching LSU nrDNA